In prokaryotic organisms, the protein synthesis, also referred to as translation, takes place on the ribosomes in the cytoplasm. In expressing recombinant DNA in prokaryotic host organisms, such as, e.g., E. coli, it is often desirable that the resultant recombinant gene product/protein should be precipitated in the cytoplasm in the form of insoluble “inclusion bodies”. After completion of fermentation and lysis of the cells, the inclusion bodies are isolated and optionally purified and the recombinant protein contained therein is solubilized by adding denaturants such as urea or guanidinium hydrochloride and maturation of said protein is accomplished by reducing the denaturing conditions. Such methods are well-known and have long been used successfully also for the industrial manufacture of recombinant proteins (cf., e.g., Lee, S. Y., Trends Biotechnol. 14 (1996) 98–105; Panda, A. K., et al., J. Biotechnol. 75 (1999) 161–172; Mattes, R., Semin. Thromb. Hemost. 27 (2001) 325–336; Clark, E. D., Curr. Opin. Biotechnol. 12 (2001) 202–207; Misawa, S., and Kumagai, I., Biopolymers 51 (1999) 297–307; and Lilie, H., Current Opinion Biotechnol. 9 (1998) 497–501).
The extent to which the proteins expressed in the cytoplasm are obtained as insoluble protein aggregates (inclusion bodies) or in soluble active or inactive form is determined essentially by the primary structure (amino acid sequence) of the protein. The primary structure of the protein determines the intrinsic biophysical/biochemical properties of the protein, said properties thus determining whether the protein will fold in the cytoplasmic environment of a microorganism to form a soluble biologically active or inactive protein or whether preferably high molecular weight protein aggregates will be formed. The folding equilibrium (formation of the soluble structured protein in relation to insoluble protein aggregates) can be influenced by the selection of the fermentation and expression conditions. For instance, by growing the prokaryotic cells at reduced cultivation temperatures and/or non-optimal induction conditions (limited inductor concentration) the recombinant plasmid-encoded protein biosynthesis rate can be reduced, thereby preventing or reducing an accumulation of the protein to form insoluble protein aggregates (cf., e.g., Kopetzki, E., et al., Mol. Gen. Genet. 216 (1989) 149–155; and EP 0 300 425). In other instances, it is desired, however, to prepare the recombinant protein via the route of inclusion bodies. Accordingly, an objective of such methods is to obtain a high yield of recombinant protein in the inclusion bodies. In this connection, it should be kept in mind, however, that other parameters, too, are of high importance in recombinant gene expression in prokaryotes, such other parameters being, for example, to achieve a recombinant gene expression as high as possible and to prevent undesired post-translational modification.